The proposed research will provide new insights into the arrangements, motions and interactions of proteins in intact cell membranes. Initial studies will be directed toward specific labeling of the anion transport protein (band 3) in the human red blood cell (rbc). Newly synthesized bifunctional spin labels will be employed in conjunction with electron paramagnetic resonance (EPR) spectroscopy to quantitate and compare the rotational motions of band 3 in intact rbc with motions in ghost membranes and ghost membranes having selected membrane skeleton proteins removed. These studies will help define protein-protein and protein-lipid interactions in rbc which determine cell shape and deformability. At the conclusion of these studies, sufficient methodology should be established to permit comparative measurements on abnormal rbc from patients with a variety of hemolytic anemias. These data may aid in establishing what structural defects are responsible for abnormal shape and increased cell fragility for a variety of rbc disorders. These same labels will be employed to label anion binding proteins which have been observed in adipocyte membranes. The transport functions of many membrane proteins in adipocytes are modulated by binding of hormones to surface receptors. Quantitation of the rotational motions of these proteins under basal and stimulated conditions will aid in understanding mechanisms of hormone action on transport activity. EPR data from the rbc and adipocyte studies will be collected using high resolution N15 spin labels. This will enable more rigorous data analysis by direct computer simulation of experimental spectra for extracting rotational diffusion coefficients and, hence, determining the states of aggregation of the labeled proteins. Spin labeled ATP analogues will be employed to examine the functional relationship between membrane bound glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and the Na+, K+-ATPase. Experiments are proposed to determine the characteristics and microenvironment of membrane associated compartments of ATP which have been observed. These studies will provide insight into the production and mode of utilization of ATP in the RBC.